Stable fibrinogen solutions and method for producing same



States atet was precluded by the federal regulatory body for biological products. 2s26533 More specifically, one of the objects of this inven- STABLE FIBRINOGEN SOLUTIONS AND METHOD tion is the provision of a method for producing a fibrino' FOR PRODUCING SAME 5 gen material wherein a conventional Cohn Fraction 1 paste is dissolved in an aqueous diluent having an ionic i gg igggf zzfigg g acg fg gfigggg2522a: strength of at least 0.30, a pH not greater than 5.85 and fomia containing a small quantity of a carbohydrate selected from the group consisting of sugars, sugar alcohols and No Drawing. application March 15, 1954 sugar a id Serial 416,447 A further object of this invention is the provision or" a dry, sterile, stable, and highly soluble fibrinogen un- 4 Clams. (Cl. 167 74) damaged by ultraviolet irradiation and freeze-drying and which can be reconstituted with water to produce a solu- This invention relates to and in general has for its tion containing at least 2 grams of fibrinogen per 100 cc. object the provision of a highly soluble, stable and sterile of solution. fibrinogen product, and to a method for producing such More specifically and by way of one: example, the product. objects of this invention may be attained by resorting to Presently, there is a serious need for fibrinogen of this the following procedure: form in connection with hypofibrinogenemia. For eX- Pooled plasma is cooled to 0 and a preliminary clariample, statistics indicate that in this country abruptio fication and precipitation is conducted using an alcoholic placentae occurs in about 0.5% of all births. Abruptio buffer of the following compositions: placentae results in the escape of thromboplastic enzymes 178 cc. of 53% ethanol at 5 1.5 cc. of acetate from the placenta into the maternal circulation, thereby buffer of pH 4.0 and ionic strength of 0.8. For each causing an immediate destruction of all of the fibrinogen liter of plasma at 0, 2.5 cc. of the alcoholic butter at in the maternal circulation, and, in turn, a massive and 5 is added and the resultant mixture is clarified by uncontrollable hemorrhage. The only known treatment continuous centrifugation at 0 to 1. for this is the actual replacement of the fibrinogen so lost Depending on the nature of the plasma, from 1.2 gms. from the blood. Hypofibrinogenemia also occurs as a to 2.3 gms. of paste is removed per liter of plasma. result of certain drastic surgery, such as thoracic and 0 Additional alcoholic buffer at 5 is then added to the pancreatic surgery whereinin some cases thromboplastins supernatant in an amount sufficient to precipitate a conenter the circulatory system, destroy the patients fibrinoventional Cohn Fraction 1 paste at an ethanol concengen and cause an uncontrollable hemorrhage. tration of 8% and ionic strength of 0.14, apH of 7.2 and Hypofibrinogenemia can also exist as an inherited cona temperature of -2.5 C. The Fraction I pasteis redition Where the patient is devoid or substantially devoid moved by continuous centrifugation at -2.5. of fibrinogen. Following this or concurrently therewith, a solvent buf- Although fibrinogen is currently available, the form in fer of the following composition is prepared and mainwhich it exists is subject to three major defects. Firstly, tained at 2 C.: solutions thereof cannot be made sufficiently stable to Withstand ultraviolet irradiation of sufficient intensity to 4 Sodium citrate .2 H O "gms-- 12.3 destroy the virus of homologous serum jaundice which is Sodium chloride gms 11.6 often associated therewith, and here it is to be noted that Citric acid .2 H O gms 2.1 this virus is largely concentrated in the fibrinogen fraction Dextrose gms derived from plasma. Secondly, the lyophilized (freeze- Distilled Water q. s ..cc 1000 dried) fibrinogen when reconstituted with water produces an insoluble residue in the amount of about five percent; The P of this Solvent buffer Should he and thirdly, it is impossible to reconstitute the dried ma- Each of Fraction 1 Paste is suspended in 3130 of terial to make up a solution of fibrinogen containing as the Solvent buffer thus P p at C: using a pi much as 2 grams of iibrinogenper 100 cc. of solution. acting mechanical and eXcilldiing f f Of A concentration of this order is particularly important in t0 the surface of the hqhld 50 that an fhiiihihg 15 P order to be able to introduce the required amount of T P 0f the Solvent buffer containliigthe DISSOh/fid fibrinogen into the circulatory system without the neces- Fraction 1 P815te is flppihXiihfiieiy and must be heihw sity f introducing mo great a volume f solution pH 5.85. The solution of Fraction I paste in the solvent Here it should be observed that it is impractical to buffer contains approximately 3 gms. of fibrinogenfor treat abruptio placentae by the transfusion of whole every 100 cc. of solution. The dissolved Fractionlpaste blood, for whole blood contains only 0.16 grams of is stirred gently and continuously, quickly warmed to 20 fibrinogen P 100 Since in cases of this kind it is and maintained at 20 for thirty minutes, and is then filnecessary to raise the fibrinogen level from 0.00 at least tered fi t through a coarse fin paper and h h h half of the normal level (0.087 gram per 100 cc. is the a tight asbestos pad (f example a pad) to Produce critical level) if clotting is to occur, simple arithmetic 6Q asparkling clear m shows that it will require the transfusion of approximately The clear fibrinogen filtrate is then immediately irra liters of Whol? blood to a wlth a normal blood diated with ultraviolet using approximately twice the involume of mars to.reach thls.level' .Even greager tensity of irradiation defined in the minimum requireamounis necessary if any fibrmogendls lost merits of the National institutes of Health, entitled Ultraz gg dunng the long'drawn'out proce me of tram 65 violet Irradiation for the Sterilization of Biological Proda! r' '1 One expedient that has been resorted to in several cases P F i g b 5 T g fig d tS lli tiOn of abruptio placentae consists of administering a fibrino- 15 lately Stem lze Halon} 5 en lzlgg gen solution containing about 0.7 gram per 100 cc. steria Pads h Sterne bulk sampkid for t e li d ith nitrogen ta d, H w th t i it f immediate determination of the clottable protein content.

the residual nitrogen mustard was so great that the manufacture and sale of fibrinogen, sterilized in this manner,

Following this, the solution is transferred under aseptic conditions and in unit dosage volumes into small vials and there lyophilized, stoppered and sealed under vacuum, all in accordance with well known practice.

When sterile water is added to reconstitute the dry material to a fibrinogen content of at least 2%, the dry material completely dissolves within five minutes and will not clot spontaneously for periods in excess of 48 hours.

In carrying out this process any of the following carbohydrates can be substituted for the dextrose added to solvent buffer in which the Fraction I paste is suspended: pentose sugars, disaccharides, sorbitol, and gluconate.

The stabilized fibrinogen of this invention, in addition to being stable to ultraviolet irradiation, when reduced to the dry state, will withstand gamma radiation of doses up to 3 X REP, without any evidence of damage. Doses of 4x10 REP, caused the destruction of 9.2% of the fibrinogen being irradiated.

The stability of reconstituted fibrinogen solutions containing 2% fibrinogen as measured by the spontaneous clotting time of the fibrinogen content thereof is indicated in the following table:

pH Sponta- Exp. N0. before Ionic Additive neous drying Strength Clotting in Hours 6.07 1 6.07 15 Dextrose, 8% 30 6. 07 15 Sorbitol, 8% 36 6.1 .15 NaGluconate, 5%.-. 65 5. 4 15 18 5. 4 25 18 6. 4 35 28 5. 4 45 63 5. 8 15 18 5. 8 25 25 53 5. 8 35 68 5. 8 .45 108 6. 05 15 34 5. 42 .45 192 From this table it will be noted that the stability of any particular solution depends on the three factors: pH, ionic strength and the carbohydrate additive used. More particularly, it is to be observed that the optimum results are obtained with a fibrinogen solution having a pH of 5.40-1.05 but at all events not greater than 5.85; having an ionic strength not less than 0.30 and containing not less than 3% of a carbohydrate additive.

A solution of fibrinogen having an ionic strength of 0.30 Will begin to precipitate at a pH value of about 5.0 as will appear from the article by Morrison, Edsall and Miller, published during 1948 in the Journal of the American Chemical Society, vol. 70. The three dimensional curve appearing in the upper left corner of page 3105 of the article Will indicate this condition where the ethanol content is zero. Thus the lower pH value contemplated herein can be expressed as that at which the fibrinogen would begin to precipitate.

Although the derivation and composition of a Cohn Fraction I solution and a Cohn Fraction I paste is well known in this field, if more detailed information concerning them is desired, reference can be made to the Cohn Patent 2,390,074 of December 4, 1945, and to the publication in the Journal of the American Chemical Society, 68, 459 (1946).

It should be noted that during 1953, over thirty human cases, in which the administration of whole blood had failed and death was virtually certain, were treated intravenously with the fibrinogen solution of this invention in doses of from 3 g. to 5 g. of fibrinogen, and recovery was complete within 2 to 3 hours.

In conclusion, it will be seen that as a result of the procedure above set forth, I have provided a dry, stable, ultra-violet irradiated fibrinogen product capable of being reconstituted and completely dissolved in water within a relatively short time for making up a solution having a sufiiciently high concentration of fibrinogen to serve as a therapeutic product.

I claim:

1. A method of preparing a stable solution of fibrinogen comprising: dissolving human fibrinogen in an aqueous diluent so that the resultant solution has an ionic strength of at least 0.30, a pH not greater than 5.85 and not less than a value at which the fibrinogen would begin to precipitate and containing at least 3% of a carbohydrate selected from the group consisting of sugars, sugar alcholos and sugar acids.

2. The method of preparing dry, sterile, human fibrinogen comprising: making up an aqueous solution of human fibrinogen; adjusting the ionic strength of such solution to at least 0.30; adjusting the pH of such solution within a range of 5.85 and such lower value at which the fibrinogen would begin to precipitate and adding to said solution at least 3% of a carbohydrate selected from the group consisting of sugars, sugar alcohols and sugar acids.

3. A dry, sterile, virus-free, stable composition of human fibrinogen containing at least 1.5 grams of a carbohydrate selected from the group consisting of sugars, sugar alcohols, sugar acids for each gram of fibrinogen, and containing an electrolyte in a quantity suflicient, when at least two grams of said composition are dissolved in cc. of water, to bring the ionic strength of the resulting solution to at least 0.30 and its pH to a value in the order of from 5.85 to such lower value at which the fibrinogen would begin to precipitate.

4. An aqueous fibrinogen solution containing not less than 3 per cent of a carbohydrate selected from the group consisting of sugars, sugar alcohols and sugar acids, said solution having an ionic strength of not less than 0.30 and a pH within the range of 5.85 to such lower value at which the fibrinogen would begin to precipitate.

OTHER REFERENCES Morrison: Proc. Soc. Exptl. Biol. and Med., vol. 78, OctoberDecember 1951, pp. 653-655. 

1. A METHOD OF PREPARING A STABLE SOLUTION OF FIBRINOGEN COMPRISING: DISSOLVING HUMAN FIBRINOGEN IN AN AQUEOUS DILUENT SO THAT THE RESULATNT SOLUTION HAS AN IONIC STRENGTH OF AT LEAST 0.30, A PH NOT GREATER THAN 5.85 AND NOT LESS THAN A VALUE AT WHICH THE FIBRINOGEN WOULD BEGIN TO PRECIPITATE AND CONTAINING AT LEAST 3% OF A CARBOHYDRATE SELECTED FROM THE GROUP CONSISTING OF SUGARS, SUGAR ALCOHOL AND SUGAR ACIDS. 